Synergistic Antiplatelet Effects of Clopidogrel and Aspirin Detected With the PFA-100 in Stroke Patients
2003; Lippincott Williams & Wilkins; Linguagem: Inglês
10.1161/01.str.0000064327.15622.e6
ISSN1524-4628
Autores Tópico(s)Venous Thromboembolism Diagnosis and Management
ResumoHomeStrokeVol. 34, No. 4Platelet Function Under Aspirin, Clopidogrel, and Both After Ischemic Stroke Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBPlatelet Function Under Aspirin, Clopidogrel, and Both After Ischemic StrokeA Case-Crossover Study Armin J. Grau, MD, PhD, Sven Reiners, Cand Med, Christoph Lichy, MD, Florian Buggle, MD and Andreas Ruf, MD, PhD Armin J. GrauArmin J. Grau From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine, Klinikum Karlsruhe (A.R.), Karlsruhe, Germany. , Sven ReinersSven Reiners From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine, Klinikum Karlsruhe (A.R.), Karlsruhe, Germany. , Christoph LichyChristoph Lichy From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine, Klinikum Karlsruhe (A.R.), Karlsruhe, Germany. , Florian BuggleFlorian Buggle From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine, Klinikum Karlsruhe (A.R.), Karlsruhe, Germany. and Andreas RufAndreas Ruf From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine, Klinikum Karlsruhe (A.R.), Karlsruhe, Germany. Originally published13 Mar 2003https://doi.org/10.1161/01.STR.0000064326.65899.ACStroke. 2003;34:849–854Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 13, 2003: Previous Version 1 AbstractBackground and Purpose— Combined antiplatelet agents may offer additive protection over single drugs after stroke. We investigated whether platelet activation is reduced under combined aspirin and clopidogrel compared with each drug alone.Methods— In a case-crossover study, 31 patients with previous atherothrombotic or lacunar stroke who were treated with aspirin (100 to 300 mg/d) received clopidogrel (75 mg/d) and both aspirin and clopidogrel for 4 weeks. Platelet function in whole blood was studied after each treatment period and in healthy control subjects to assess activation-dependent antigens CD62p and CD63 by flow cytometry and collagen/epinephrine (CEPI-CT) and collagen/ADP (CADP-CT) closure times with the platelet function analyzer PFA-100, which investigates platelet-related function under shear stress.Results— CD62p expression and CD63 expression were not different under the 3 treatment regimens. CD63 but not CD62p expression was lower in control subjects than in stroke patients regardless of the antiplatelet treatment (P<0.05). CEPI-CT was prolonged under aspirin and aspirin plus clopidogrel compared with clopidogrel monotherapy (P<0.0001). CADP-CT was longer under combination therapy than under aspirin (P=0.0009) or clopidogrel (P=0.0074) or in control subjects (P=0.0010), mainly because of strong prolongation in a patient subgroup (28%).Conclusions— CD63 expression reflecting the release of platelet lysosomes is consistently increased after stroke and incompletely suppressed by treatment with aspirin, clopidogrel, or both. The strong prolongation of CADP-CT under combined aspirin and clopidogrel in a patient subgroup may indicate a lower risk of thrombosis but also a higher risk of hemorrhage. The predictive value of platelet activation parameters requires investigation in prospective studies.Antiplatelet drugs are the treatment of choice for secondary prevention after cerebral ischemia of noncardioembolic origin. However, the efficacy of presently available drugs is limited. Compared with placebo, aspirin reduced the relative risk of vascular events by 13% according to two meta-analyses1,2 and stroke risk by 18% in the European Stroke Prevention Study 2.3 Under clopidogrel, the relative risk of a compound outcome cluster of stroke, myocardial infarction, and vascular death was marginally, although significantly, lower than under aspirin (−8.4%), whereas the risk reduction for stroke alone was not significant.4 Aspirin inhibits platelet aggregation by inhibition of cyclooxygenase, whereas clopidogrel reduces platelet activation via ADP receptor-dependent pathways. On the basis of these different modes of action, it is an attractive concept that the combination of both drugs may have additive effects on plateletSee Editorial Comment, page 854inhibition. The combination of aspirin and clopidogrel or ticlopidine was shown to be a successful treatment strategy after coronary stenting and in unstable angina pectoris5,6 and is currently being tested in a large, randomized, placebo-controlled study on secondary prevention after stroke. However, it is unknown at present whether the combination of aspirin and clopidogrel leads to reduced platelet activation in patients at high risk for ischemic stroke or other vascular events.We performed a case-crossover study to test the hypothesis that in patients with a history of stroke, platelet activation parameters are reduced under combination therapy with aspirin and clopidogrel compared with either therapy alone. We also investigated inflammatory parameters under treatment with aspirin, clopidogrel, and both because aspirin but not clopidogrel possesses anti-inflammatory potential and inflammatory indexes were shown to predict the risk of vascular events.7 Furthermore, we compared platelet activation and inflammatory parameters in patients with previous stroke and healthy control subjects. Platelet function in whole blood was assessed by flow cytometry and by the platelet function analyzer (PFA-100).Subjects and MethodsIn the present study, patients who had suffered ischemic stroke and were treated with aspirin for secondary prevention received clopidogrel alone or a combination of clopidogrel and aspirin for 4 weeks. Platelet function was determined at the end of each treatment period and included flow cytometric assessment of the expression of p-selectin (CD62p) or lysosome-associated membrane protein (CD63) and studies with the PFA-100 (Dade Behring) assessing collagen/epinephrine (CEPI) and collagen/ADP (CADP) closure time (CT).Patients between 18 and 80 years of age were eligible for study participation if they had suffered ischemic stroke between 6 months and 2 years ago, if the index stroke was caused by large-artery atherosclerosis or cerebral microangiography, and if they had received aspirin (100 to 300 mg/d) for at least 3 months. Large-artery atherosclerosis was diagnosed by duplex studies or angiography showing stenoses of brain-supplying arteries of ≥50% diameter reduction and a typical morphology for atherosclerotic lesions. Cerebral microangiopathy was diagnosed if neuroimaging showed ischemic lesions of 193 seconds and CADP-CT >133 seconds were considered prolonged. Measurements were performed in duplicate. The intratest variability was 0.050 for CEPI and 0.085 for CADP cartridges. CRP was determined by immunoturbidimetric assay (Aeroset; threshold of detectability: 3 mg/L). Fibrinogen was measured by functional coagulation testing (derived fibrinogen) with Recombiplastin (Instrumentation Laboratory) as reagent. Leukocyte counts were assessed by Coulter counter analysis.Data are presented as mean and SD or median and percentiles as appropriate. We used the Friedman test for intraindividual comparisons between the 3 treatment regimens. For comparisons of the 4 platelet function parameters between treatment groups, differences were considered significant at P≤0.0125. The Wilcoxon signed-rank test was applied for further analyses of treatment regimens if the Friedman test yielded significant results. The Mann-Whitney U test was used to compare patients and control subjects; the Spearman rank correlation coefficient was used to correlate parameters; and Fisher's exact test was used for analyses of categorical variables. Analysis of variance (ANOVA) was applied to analyze the influence of various factors on a parameter. Variables without normal distribution received logarithmic transformation before ANOVA. We used the software package SAS (version 8.02) for the analyses.ResultsNone of the 31 stroke patients had symptoms attributable to cerebral ischemia or developed any cerebral or systemic bleeding during the study. One patient withdrew from the study during therapy with aspirin plus clopidogrel because of sudden severe shoulder pain. Bleeding into the shoulder joint was excluded by MRI. One patient was excluded shortly after initiation of the combination therapy because of a first epileptic seizure. Therefore, 29 patients completed the entire study.Under the 3 treatment regimens, the expression of CD62p and CD63 was not significantly different (Table 2). In contrast, CEPI-CT and CADP-CT showed significant differences between treatments (P<0.0001, respectively; Table 2, Figures 1 and 2). Regarding CEPI-CT, differences were due to prolonged clot formation under aspirin (P<0.0001) and aspirin plus clopidogrel (P 300 seconds) in 23 of 31 patients (74.2%) under aspirin and in 26 of 29 patients (89.7%) under both aspirin and clopidogrel (P=0.18). Normal values (≤193 seconds) were found in 5 of 31 patients (16.1%) despite medication with aspirin and in 1 of 29 patients under the combination therapy (3.4%; P=0.20). The CADP-CT was prolonged under the combination therapy compared with monotherapy with either aspirin (P=0.0009) or clopidogrel (P=0.0074). Results under aspirin and clopidogrel were not different (P=0.51). The CADP-CT was outside the normal range (>133 seconds) in 2 patients (6.5%) under clopidogrel but in 8 patients (27.6%) under the combination therapy (P=0.039). Complete inhibition of CADP-CT (>300 seconds) occurred in 7 patients under the combination therapy but in 0 of the patients under clopidogrel (P=0.004). CEPI-CT and CADP-CT were correlated with each other under clopidogrel (R=0.32, P=0.006) and the combination therapy (R=0.54, P 0.53, P<0.001). CEPI-CT and CADP-CT on one side and CD62p and CD63 expression on the other side were not significantly correlated with each other. TABLE 2. Platelet Activation Parameters Under Different Treatment Regimens in Patients With a History of Ischemic StrokeParameterTreatment RegimensP Value*AspirinClopidogrelAspirin+ClopidogrelData are given as median and 25 to 75 percentile.*P values by Friedmann test; in order to adjust for multiple tests, P values 300120>300(270–>300)(106–148)(>300–>300)<0.0001Collagen-ADP closure time(s)8689100(72–94)(76–105)(88–196) 0.30) or between patients with lacunar stroke and patients with stroke resulting from large-artery atherosclerosis (P>0.25). Smoking, hypertension, hypercholesterolemia, diabetes mellitus, and the presence of coronary heart disease and/or peripheral arterial disease did neither influence CEPI-CT, CADP-CT, or CD63 expression. Diabetes mellitus (P=0.003) and hypercholesterolemia (P=0.010) were associated with higher CD62p expression (ANOVA). Leukocyte counts, platelet counts, and CRP and fibrinogen levels were not different under the 3 treatment regimens (P>0.10).Control subjects had lower CD63 expression (median, 1.0%; 25th and 75th quartiles, 0.2% and 2.8%) than patients under all 3 therapeutic regimens (P<0.05). In contrast, there was no difference regarding CD62p expression (control subjects: median, 2.3%; 25th and 75th quartiles, 0.7% and 3.6%). The CEPI-CT was similar in patients under treatment with clopidogrel and control subjects (median, 117 seconds; 25th and 75th quartiles, 108 and 127 seconds). The CADP-CT (median, 83; 25th and 75th quartiles, 81 and 93 seconds) was not different from patients treated with aspirin or clopidogrel; however, it was shorter than in patients under aspirin plus clopidogrel (P=0.001). Leukocyte counts were 5.17±1.40×109/L compared with 6.77±1.70×109/L (P=0.003), and fibrinogen levels tended to be lower in control subjects than in patients under therapy with aspirin (2.97±0.65 versus 3.50±1.06 g/L; P=0.069), whereas no difference existed regarding platelet counts and CRP levels.DiscussionCombination therapy with aspirin and clopidogrel was well tolerated and safe in our patients during the short treatment period of 4 weeks. Both serious adverse events that occurred were not causally related to the study medication. The main hypothesis of the present investigation was that in patients after stroke, aspirin plus clopidogrel may reduce platelet activation to below the level reached with each single agent alone. This study shows that the CADP-CT assessed with the PFA-100 was prolonged under combination therapy, whereas the 3 other platelet activation markers were not significantly changed.We chose flow cytometry and the PFA-100 because both methods allow platelet function analysis in whole blood.12 Aggregometry with platelet-rich plasma and measurement of platelet release products have the problem of potential artifactual platelet activation during blood sampling, and release products may lack sensitivity because of dilution effects in plasma. Flow cytometric analysis of platelets is a relatively new and powerful technique that has been developing into a useful clinical tool.12 It has the advantage of little artifactual in vitro activation, particularly because blood fixation was used in our study. Previous studies showed that CD62p and CD63 expression by platelets is increased in acute ischemic stroke.9,13 CD62p (p-selectin) is a glycoprotein localized on the α-granule membrane that is rapidly translocated to the cell surface after stimulation and mediates the adhesion of platelets to leukocytes. P-selectin upregulates tissue factor in monocytes, promotes fibrin deposition, and leads to leukocyte accumulation in areas of vascular injury associated with thrombosis and inflammation.14,15 CD63 is expressed on the platelet surface after the release of lysosomes. It may protect the plasma membrane against degradation by lysosomal proteins; however, its biological function is not sufficiently understood.16 In a baboon model, degranulated platelets shed CD62p from the cell surface but continued to circulate and function.17 Such shedding after cell activation may explain why CD62p expression was not increased in our patients in the chronic stage after stroke. In contrast, CD63, for which shedding is not known to occur, was expressed more strongly in patients than control subjects.In our study, neither clopidogrel nor the combination of aspirin and clopidogrel led to lower CD63 expression than therapy with aspirin. These results indicate that currently available antiplatelet drugs, even if combined, do not inhibit the release of platelet lysosomes in vivo, a mechanism that requires strong platelet activation.18 In patients with previous myocardial infarction, clopidogrel with and without aspirin suppressed CD62p and CD63 expression after stimulation with ADP or thrombin.19 Assessment of drug effects on platelet reactivity in vitro was not performed in our study but may be a meritorious focus of future research.The PFA-100 assesses platelet-related function, in particular adhesion and aggregation, under shear stress. Its advantages are ease of operation, rapidity, and comparability between different laboratories. It was shown to reliably monitor platelet function under treatment with aspirin and GPIIb/IIIa receptor antagonists and to be useful in screening for congenital and acquired platelet dysfunction.10–12,20–23 It is not sensitive to heparin or defects in coagulation proteins, eg, hemophilia A.12,21 To the best of our knowledge, the PFA-100 has not been applied in clinical stroke. As expected, the CEPI-CT was strongly prolonged under treatment with aspirin compared with clopidogrel or the control group. Despite intake of aspirin, 16.1% of our stroke patients had normal CEPI-CT values. In a PFA-100–based study using the same cutoff value, the rate of "aspirin resistance" was 9.5% in patients with stable cardiovascular disease receiving 325 mg/d aspirin.11 Measuring platelet aggregation in platelet-rich plasma, Helgason et al24 detected aspirin resistance in 20.6% of their stroke patients taking ≤325 mg/d aspirin. Aspirin resistance defined by in vivo thromboxane biosynthesis was recently shown to predict ischemic events.25 Patients with normal CEPI-CT values despite aspirin medication may be at increased risk of ischemic events; however, prospective data based on the PFA-100 are not yet available.Clopidogrel acts via inhibition of ADP-induced platelet activation. In accordance with previous studies,9 treatment with clopidogrel was not reflected by prolonged CADP-CT. This shows that the current setup of the PFA-100, mainly the high ADP concentration in CADP cartridges, is not suitable for monitoring therapy with clopidogrel or ticlopidine. Most interestingly, CADP-CT was longer under combined aspirin and clopidogrel than under either monotherapy. In patients undergoing percutaneous transluminal coronary angioplasty, the combination of ticlopidine and aspirin led to a nonsignificant increase in CADP-CT compared with aspirin alone21 or was associated with normal CADP-CT23; however, ticlopidine medication was shorter than in our study. The combination therapy was associated with a heterogenous response among our patients. About one fourth of the subjects showed a very strong inhibitory effect, whereas in the other patients, the difference from both monotherapies was not significant. In the subjects who showed a strong inhibition, the combination therapy could be associated with good protection against ischemic events but also with an increased bleeding risk. It is unknown why some individuals react with a strong response to combined aspirin and clopidogrel and others do not. It was recently shown that closure times in the PFA-100 are highly dependent on the von Willebrand factor.26 Differences in von Willebrand factor levels could possibly explain variations between patients. It is a limitation of our study that the von Willebrand factor was not assessed. Furthermore, the nonrandomized, non–placebo-controlled sequential treatment design could be regarded as a limitation, but it is unlikely that it affects the data interpretation. Similarly, it is unlikely that effects seen under the combination therapy represent late treatment effects of clopidogrel rather than effects of the combination therapy.Aspirin but not clopidogrel possesses an anti-inflammatory mode of action; however, the 3 inflammatory parameters assessed were not different under the medication with or without aspirin. In accordance with previous studies,27 leukocyte counts and fibrinogen levels were higher after stroke than in healthy control subjects, although the difference was not significant for fibrinogen. We did not use a high-sensitivity test for CRP assessment and may therefore have overlooked small differences in CRP.From the results of this study, it appears meritorious to investigate in larger studies whether the CADP-CT is a predictor of ischemic events or bleeding complications under treatment with combined aspirin and clopidogrel and whether normal CEPI-CT predicts an increased rate of ischemic events under monotherapy with aspirin. CD62p and CD63 expression may not be suitable for monitoring therapy with aspirin, clopidogrel, or both after stroke. Although not influenced by the antiplatelet agents studied here, CD63 expression could still be a predictor of the risk of ischemic events, an issue that requires investigation in a larger prospective study.This study was supported by a grant from Sanofi-Synthelabo Inc. Dade-Behring Inc financially supported our measurements with the PFA-100. We wish to thank Dagmar Oehmichen for her valuable technical assistance.FootnotesCorrespondence to Armin J Grau, MD, PhD, Neurologische Klinik, Städtisches Klinikum Ludwigshafen a.Rh., Bremserstr. 79, D-67063 Ludwigshafen, Germany. E-mail [email protected] References 1 Barnett HJM, Eliasziw M, Meldrum HE. Drugs and surgery in the prevention of ischemic stroke. N Engl J Med. 1995; 332: 238–248.CrossrefMedlineGoogle Scholar2 Algra A, van Gijn J. Aspirin at any dose above 30 mg offers only modest protection after cerebral ischemia. 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